The present invention relates to a radioimmunoassay involving Australia antigen. The latter has been renamed over the past years; such names as hepatitis associated antigen (HAA), serum hepatitis antigen (SH), hepatitis B antigen and hepatitis B surface antigen (HB.sub.s A.sub.g) are recognized as the same by experts in the field. Throughout this disclosure, Australia antigen will be referred to as HB.sub.s A.sub.g, which is generally accepted worldwide.
The present assays for HB.sub.s A.sub.g which are most widely used because of their sensitivity and accuracy are the radio-immunoassays. The current ones available make use of a substrate, usually a plastic bead, on to which is very likely to be attached an IgG (immunoglobulin) fraction of anti-HB.sub.s antiserum. The latter is either from a human donor or from immunized animals such as rabbits, guinea pigs, horses, sheep, goats, etc. This IgG fraction is composed of several antibodies which include the antibody to HB.sub.s A.sub.g (anti-HB.sub.s). Other antibodies which may be expected in such antisera are antibodies to other agents to which the animal was exposed. For example, humans may be expected to have antibodies to every agent to which they were ever exposed. These may include polio, measles, pertussis, diphtheria and tetanus toxoids, for example. Therefore the IgG fraction of any antiserum should be expected to contain a variable amount of specific antibody to HB.sub.s A.sub.g, and that amount being probably no more than 1% of the total globulin fraction. Thus, with some prior art assay reagents, the HB.sub.s A.sub.g in a test sample will combine with the small amount of anti-HB.sub.s on the plastic bead. A radioactively labeled anti-HB.sub.s (labeled with iodine 125) is next added to combine with the HB.sub.s A.sub.g forming a sandwich of anti-HB.sub.s - HB.sub.s A.sub.g -labeled anti-HB.sub.s. This bead is then placed in a gamma counter to measure the amount of radioactivity. The greater the amount of radioactivity relevant to controls (normal human sera), the greater the amount of HB.sub.s A.sub.g. Assays such as the one described above have been described in publications (Purcell, R. H., Gerin, J. L., Almeida, J. B., and Holland, P. V., Intervirology 2: 231-243 (1973/74) and Purcell, R. H., Wong, D. C., Alter, H. J., and Holland, P. V., Appl. Microbiol. 26:478-484 (1973).
However, in order to have any reasonable chance of getting any really good sensitivity in a test along the above lines, "dissociated" anti-HB.sub.s must be made and used either as the radioactive labeled anti-HB.sub.s, or for use instead of the IgG fraction to initially coat the solid substrate involved.
Unfortunately, the making of "dissociated" antibody for such a purpose is quite difficult and expensive, so that its making and use adds greatly to the total expense involved in any radioimmunoassay for hepatitis B which uses it thus.
"Dissociated" antibody is made by reacting purified HB.sub.s A.sub.g with the IgG fraction containing anti-HB.sub.s, washing the complex, and then dissociating or separating the anti-HB.sub.s from the HB.sub.s A.sub.g. There are several ways of accomplishing this and the one preferred today is known as affinity chromatography. Purified HB.sub.s A.sub.g is attached to some solid material such as Sepharose B (Pharmacia) resin which is poured into a column. The IgG fraction containing anti-HB.sub.s is allowed to filter through. Anti-HB.sub.s reacts and combines with HB.sub.s A.sub.g. The column is washed and sodium thiosulfate or some other known reagent for dissociation of antigen-antibody complexes is added. The anti-HB.sub.s fraction is recovered, dialyzed and concentrated and can then be used.
"Dissociated" antibody is difficult to produce with any consistency and the process is expensive. Problems usually arise because of low yields (due to incomplete dissociation) and yields of anti-HB.sub.s with low combining power (avidity). The reason for the latter in unknown but may be due to incomplete uncovering of all anti-HB.sub.s combining sites or denaturation or both.
The present invention provides a better reagent which is sensitive and at the same time relatively inexpensive to produce.
The present invention secures high sensitivity without the great added expense of making up "dissociated" anti-HB.sub.s separately. It does this by making a solid phase reagent in which the substrate has two coats or layers, the first of which is purified HB.sub.s A.sub.g and the second of which is anti-HB.sub.s which has been contacted with the HB.sub.s A.sub.g in no very purified form. In other words the second coat is not a separately made "dissociated" form of anti-HB.sub.s, but an ordinary source which contains anti-HB.sub.s as part of a very impure material, such as (especially) antiserum containing anti-HB.sub.s or an IgG fraction of antiserum containing anti-HB.sub.s.
The first coat, the purified HB.sub.s A.sub.g, is applied on the substrate to an extent which permits its complete coverage by anti-HB.sub.s. The second coat, which comprises anti-HB.sub.s, must be applied in an quantity which completely covers the HB.sub.s A.sub.g, occupying substantially completely the reacting sites of the HB.sub.s A.sub.g, and includes an excess of antibody reacting sites which remain free.
The HB.sub.s A.sub.g coat is allowed to select, by specific reaction, the antibody, anti-HB.sub.s. Therefore, a second coat or layer results which is exclusively specific antibody to HB.sub.s A.sub.g. Since the second coat, like other antibodies used in such assays, has molecules each with two reactive sites, and a suitable excess of the anti-HB.sub.s has been applied, some of the antibody binding sites are available for further reaction, so that, the anti-HB.sub.s can combine with HB.sub.s A.sub.g in test samples. In effect the reagent of the invention is highly specific because the antibody comprising the second coat is specific antibody capable of reacting with HB.sub.s A.sub.g and only HB.sub.s A.sub.g.
A purpose of the present invention is to provide a highly sensitive solid state HB.sub.s A.sub.g assay whose expense will be greatly reduced.
Describing the present invention somewhat more specifically, a polystyrene bead or other appropriate substrate is first coated with purified HB.sub.s A.sub.g. The extent of purification required will be discussed further hereinbelow. In making this coating, care should be taken that it be done only to an extent which is capable later of being entirely covered with anti-HB.sub.s.
Then after washing, the antigen-coated bead is allowed to react with a source of anti-HB.sub.s, to completely cover the HB.sub.s A.sub.g with the anti-HB.sub.s, and also to provide on the bead an excess of antibody reacting groups which remain free.
After washing, there is thus secured a reagent in the form of a doubly coated bead or other substrate whose under-coating layer is HB.sub.s A.sub.g limited in extent to that which can be completely covered with anti-HB.sub.s, and whose outermost layer is a complete covering made up of anti-HB.sub.s and that alone, and in an amount which includes an excess of antibody reacting groups which remain free.
Once this reagent is made, one can use it for detecting HB.sub.s A.sub.g in a test sample, such as serum. This is done as follows:
For detecting hepatitis antigen (HB.sub.s A.sub.g) in serum, the reagent and test serum as to which the possible presence of HB.sub.s A.sub.g is desired to be tested, are brought together and if there is HB.sub.s A.sub.g in the serum, HB.sub.s A.sub.g from the serum will react with free antibody reacting groups on the reagent to attach itself to the outermost coat of the reagent consisting of antibody to hepatitis surface antigen.
After washing, radioactively labeled antibody will be added to react with any such attached HB.sub.s A.sub.g from the test serum. Any excess of radioactively labeled antibody beyond what has thus reacted is then removed and the presence or absence of significant radioactivity in what remains will determine the presence or absence of HB.sub.s A.sub.g in the test serum, within the limits of sensitivity of the particular test.
This present process has been found to have very good sensitivity, as tests results tabulated hereinbelow with show.
Thus, the present invention involves a sensitive solid state process in which the presence of HB.sub.s A.sub.g in the test serum can be determined without ever at any stage requiring the making of separate "dissociated" antibody in some separate process of manufacture, which latter is a very expensive part of all the previous processes, if good sensitivity is to be achieved. For this reason, this present process can be expected to be especially economical and inexpensive in its totality, as compared to any previous solid state process which might secure comparable sensitivity.